Primer, kit and identification method for identifying strong and weak duck plague strains

Abstract

The present invention discloses a primer, a kit and an identification method thereof for identifying strong and weak duck plague strains. The present invention splices the X fragment with the 5'UTR region of the weak strain C-KCE, and designs identification primers as shown in SEQ ID NO.1 and 2 based on the spliced ​​C-KCE LORF5 5'UTR region. Compared with the current national standard method for detecting duck plague (which cannot distinguish between strong and weak duck plague), the PCR method for identifying strong and weak duck plague strains established by the present invention based on the spliced ​​C-KCE LORF5 5'UTR region can effectively and accurately identify strong and weak duck plague strains, and can detect a minimum of 0.46pg of strong DPV and 46pg of weak DPV.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer, a kit and an identification method for identifying strong and weak duck plague strains. Background Art

[0002] Duck plague (DP), also known as duck viral enteritis (DVE), is an acute, febrile, septicemic infectious disease caused by duck plague virus (DPV). After DPV infection, the clinical manifestations of sick ducks are body temperature rising above 43°C, eyelid edema and adhesion, nasal discharge, diarrhea and discharge of gray-white loose feces, and some sick ducks have swollen heads, so it is also called "big head plague". Acute septicemia is the main feature of sick duck dissection. The skin, mucosa, serosa and internal organs of sick ducks show varying degrees of bleeding; the esophageal mucosa is covered with gray-yellow pseudomembranes and a small number of bleeding spots. Bleeding bands often appear in the esophageal dilatation, glandular stomach junction and duodenum, and congestion in the duodenum and rectum is more serious. Histopathological observations of sick ducks also show serious lesions in lymphoid organs and digestive organs, a decrease in the number of lymphocytes in central immune organs, swelling and shedding of intestinal mucosal epithelial cells, and hyperplasia of connective tissue in the intestinal lamina propria. Since duck plague was reported in the Netherlands in 1923, it has been found in duck farming all over the world. Huang Yinxian first reported the discovery of duck plague in my country in 1957, and described in the article that the disease had existed in Guangdong Province before 1949, and was subsequently found in all regions of the country. The disease has a wide range of prevalence, high morbidity and mortality, and seriously threatens the development of my country's duck farming industry.

[0003] One of the epidemiological characteristics of duck plague is that the latent infection of DPV appears clinically healthy, which can cause an outbreak of duck plague when conditions are right. Shawky et al. first confirmed that the main site of DPV latency is the trigeminal ganglion, and DPV can also be detected in a few lymphocytes. Survivor ducks can still excrete toxins to the outside world 4 years after infection. Under conditions such as reduced host immunity, the virus lurking in the body of the surviving duck can be reactivated, leading to a secondary outbreak and epidemic of duck plague. Therefore, how to diagnose and identify duck plague virus is crucial.

[0004] The duck plague virus genome is huge, with a total of 78 ORFs and a size of about 160kb. The whole genome nucleotide sequences of different DPV strains are highly similar, but there are sequence differences between strong and weak strains at some ORFs. Based on the sequence differences between strong and weak strains of duck plague at the UL2 gene, Xie L et al. compared the UL2 gene sequences of 10 strong DPV strains and 11 weak duck plague strains and found that the weak duck plague strains all lacked the 528bp UL2 gene B fragment, and established PCR and fluorescence quantitative PCR methods that can theoretically specifically detect strong duck plague strains. However, the results showed that the two established methods were positive for the detection results of strong duck plague strains (Yulin2016-30D strain) and 5 weak duck plague strains, indicating that it is not possible to establish a specific detection or differentiation method for strong and weak duck plague strains based on the UL2 gene B fragment. At the same time, the current national standard method for detecting duck plague cannot effectively distinguish strong and weak duck plague strains. Therefore, it is urgent to establish an identification method that can distinguish strong and weak duck plague strains. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a primer, a kit and an identification method thereof for identifying strong and weak duck plague strains. The PCR method for identifying strong and weak duck plague strains based on the spliced ​​DPV C-KCE LORF5 5'UTR region can effectively and accurately identify strong and weak duck plague strains, and can detect a minimum of 0.46pg of strong DPV and 46pg of weak DPV.

[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] A primer for identifying strong and weak duck plague strains, the sequences of which are shown in SEQ ID NOs. 1 and 2. The specific sequences are:

[0008] Strong and weak virus identification-F: GATGAATCTCTCCGCCAT (SEQ ID NO.1)

[0009] Strong and weak toxicity identification-R: GTACGTGGTGTAGCCGAAT (SEQ ID NO.2)

[0010] A kit for identifying strong and weak duck plague strains, comprising the above primers.

[0011] Furthermore, the test kit is used to identify or assist in identifying the virus to be tested as a strong strain or a weak strain of duck plague, or to detect whether the sample to be tested is infected with a strong strain or a weak strain of duck plague virus.

[0012] A method for identifying a virulent strain of duck plague for non-diagnostic or therapeutic purposes comprises the following steps:

[0013] (1) Extracting DNA from the sample to be tested;

[0014] (2) Using the combination described in claim 1 or the kit described in claim 2 to perform PCR detection, and judging whether it is a strong strain or a weak strain sample based on the size of the amplified target fragment.

[0015] Furthermore, the DNA of the sample to be tested comes from a cotton swab sample.

[0016] Furthermore, the PCR reaction system included 5 μL of 2×Rapid Taq Master Mix, 1 μL of 10 ng / μL template, and the concentrations of upstream and downstream primers were 0.4 μmol / L, respectively, and finally ddH2O was added to make up to 10 μL.

[0017] Furthermore, the PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s; annealing at 55°C for 15 s; extension at 72°C for 45 s, for a total of 35 cycles; and finally extension at 72°C for 5 min.

[0018] Furthermore, in step (2), if the amplified fragment is 2454 bp, it is a strong strain sample; if the amplified fragment is 536 bp, it is a weak strain sample.

[0019] Beneficial effects of the present invention:

[0020] The current national standard method for duck plague detection (GB / T 22332-2008) cannot distinguish between strong and weak strains of duck plague, and relies on tissue or blood samples. Sampling has a great impact on the production performance of animals, has high requirements for collection technology, and has a complex DNA extraction procedure. Cotton swab samples have the advantages of convenient and rapid collection, low irritation to animals, simple operation, and easy to be mastered by breeding personnel in production. It is an ideal sample collection method for duck plague diagnosis and detection. The PCR method for distinguishing strong and weak strains of duck plague based on the C-KCE LORF5 5'UTR region after duck plague splicing established by the present invention can effectively and accurately identify strong and weak strains of duck plague in cotton swab samples, and can detect a minimum of 0.46pg of strong DPV and 46pg of weak DPV, providing technical support for the rapid differential diagnosis of strong and weak DPV strains and epidemiological investigations. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the sequence alignment result of 5'UTR region of LORF5 gene of 32 DPV strains;

[0022] Figure 2 The results of X-fragment electrophoresis detection;

[0023] Figure 3 For identification of strong and weak toxins - F / R primer electrophoresis test results;

[0024] Figure 4 The results of DPV CHv template primer concentration optimization;

[0025] Figure 5 Optimize the number of cycles for the DPV CHv template;

[0026] Figure 6 Optimization results for DPV CHv annealing temperature;

[0027] Figure 7 The results of primer concentration optimization for DPV CVCC AV1222 template;

[0028] Figure 8 Optimized results for the DPV CVCC AV1222 template cycle times;

[0029] Fig. 9 Optimization results of annealing temperature for DPV CVCC AV1222 template;

[0030] Fig.10 This is the test result of mixed template of strong and weak duck plague virus;

[0031] Fig.11 For identification of strong and weak toxins - F / R primer specificity test results;

[0032] Fig.12 It is the result of DPV CHv sensitivity test;

[0033] Fig.13 It is the result of DPV CVCC AV1222 sensitivity test;

[0034] Fig.14 The results of the sensitivity test of the national standard PCR method to DPV CHv;

[0035] Fig.15 The results of the sensitivity test of the national standard PCR method to DPV CVCC AV1222;

[0036] Fig.16 This is the oral swab test result of the DPV CHv group;

[0037] Fig.17 The results of cloacal swab test in the DPV CHv group;

[0038] Fig.18 This is the oral swab test result of the duck plague vaccine immunization group;

[0039] Fig.19 This is the cloaca swab test result of the duck plague vaccine immunization group;

[0040] Fig. 20The results of PCR test for strong and weak virus identification in cloacal swabs of Mianyang duck farm;

[0041] Fig.21 This is the national standard PCR test result of cloacal swabs in Mianyang duck farm;

[0042] Fig. 22 The PCR test results for strong and weak virus identification in cloacal swabs of Ya'an duck farm;

[0043] Fig.23 This is the national standard PCR test result of cloacal swabs in Ya'an duck farm;

[0044] Fig.24 The results of PCR test for strong and weak virus identification of cloacal swabs from No. 1 Qionglai Duck Farm;

[0045] Fig.25 This is the national standard PCR test result of cloacal swabs from No. 1 Qionglai Duck Farm;

[0046] Fig.26 The results of PCR test for strong and weak virus identification of cloacal swabs from Qionglai Duck Farm No. 2;

[0047] Fig. 27 These are the national standard PCR test results of the cloaca swabs from Qionglai Duck Farm No. 2. DETAILED DESCRIPTION

[0048] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0049] Example 1 Primer Design

[0050] The 5'UTR region sequences of the LORF5 gene of 27 strong and 5 weak duck plague strains prevalent and isolated in my country were obtained from NCBI GenBank (Table 2). NCBI Blast revealed that there was a large difference in the sequence length of strong and weak DPV strains in this region. Based on this difference, primers LORF5-F / R were designed to identify strong and weak duck plague strains ( Figure 1When the primers were used to amplify the DNA of the attenuated duck plague commercial vaccine (CVC AV1222) and the attenuated duck plague Cha strain stored in our laboratory, it was found that in addition to the expected band of about 394 bp, a non-target band of about 2000 bp (X fragment) was also produced after PCR amplification. This X fragment was recovered for sequencing and splicing, and it was found that the X fragment sequences of the CHa strain and the CVC AV1222 strain were highly consistent with the sequences of the DPV virulent strain and the VAC clone03 strain in this region (such as Figure 2 shown).

[0051] Based on this, it is speculated that there are two DPV attenuated templates in the existing commercial vaccine CVCC AV1222 and the attenuated strain CHa: one template sequence is consistent with the NCBI uploaded sequence; the other template has an additional "X fragment". The possible reason for the existence of two fragments is speculated that the fragments of the DPV strong virus may be missing to varying degrees during the attenuation process of the strong virus, resulting in the existence of two templates; or as the number of generations in cells or chicken embryos increases, the DPV attenuated strain has undergone fragment recombination, replacement or mutation. However, the challenge protection experiment of ducks immunized with the current commercial vaccine CVCC AV1222 strain showed that the AV1222 strain can still protect against 100LD 50 The ducks challenged with DPV provided complete protection, indicating that the presence of X segment may not cause the vaccine strain to revert to virulence and change in immunogenicity, and the X segment may have little correlation with the virulence of DPV.

[0052] Considering that the attenuated vaccine commonly used in farms in my country is AV1222 strain (C-KCE), in order to ensure the sensitivity and specificity of the established PCR method, this application redesigned a pair of identification primers (strong and weak toxin identification-F / R) that can distinguish between strong and weak duck plague toxins based on the spliced ​​C-KCE LORF5 5'UTR region ( Figure 1 ), the national standard-F / R primer sequence and detection method refer to the national standard method for duck plague diagnosis in my country (GB / T 22332-2008), and the primer sequence is shown in Table 1.

[0053] Table 1 Primer sequences

[0054]

[0055] Table 2 Information of 32 DPV strains

[0056]

[0057]

[0058] Example 2 Primer feasibility verification

[0059] Using DPV CVCC AV1222, DPV CHa and DPV CHv genomes as templates, PCR amplification was performed using primers LORF5-F / R, and a blank control was set up. 5 μL of PCR product was taken for agarose gel electrophoresis. Referring to the instructions of 2×Rapid TaqMaster Mix (Vazyme, catalog number: P222-02), the total volume of the PCR reaction was 10 μL, including 2×Rapid TaqMaster Mix 5 μL, 0.4 μmol / L of each upstream and downstream primer, 1 μL of template (concentration of 10 ng / μL), and ddH2O was added to 10 μL. The PCR reaction program was set as follows: 95℃3min; 95℃15s; 55℃15s; 72℃45s; 30 cycles; 72℃5min. 5 μL of PCR product was taken for agarose gel electrophoresis to detect whether the product size was correct and to confirm whether there were primer dimers.

[0060] The electrophoresis results showed that the expected band of 3698 bp could be amplified using the DPV CHv genome as a template ( Figure 2 , lane 3); using DPV CVCC AV1222 and DPV CHa genomes as templates, a 394 bp band and a 2000 bp band were amplified ( Figure 2 , Lanes 1 and 2). The band of about 2000 bp is an unexpected target band (fragment X). To determine the reason why the primers amplify the attenuated template to produce fragment X, the amplified products of lanes 1 and 2 were purified and sent to Shanghai Bioengineering Co., Ltd. for sequencing.

[0061] The sequencing results were spliced ​​together, and it was found that the X fragment was highly consistent with the sequences of the DPV strong and VAC clone03 strains. The X fragment obtained by sequencing was spliced ​​with the 5'UTR region of the weak strain C-KCE, and a pair of strong and weak toxicity identification-F / R primers were redesigned based on the spliced ​​C-KCE LORF5 5'UTR region (SEQ ID NO.7) Figure 2 ), the primer feasibility test results showed that the expected band of 536 bp could be amplified using DPV CVCCAV1222 and DPV CHa genome as templates ( Figure 3 Lanes 1 and 2) showed that the expected band of 2454 bp was amplified using DPV CHv as a template (Lane 3, Figure 3), indicating that this pair of primers can be used to distinguish between the strong strain of DPV CHv and the weak strains of DPV CVCC AV1222 and DPV CHa.

[0062] Example 3 Optimization and determination of PCR reaction conditions

[0063] 1. Using duck plague virulent DNA as template (10ng / μL), optimize the primer concentration

[0064] The total volume of the PCR reaction was 10 μL, including 5 μL of 2× Rapid Taq Master Mix, 1 μL of template (concentration was 10 ng / μL), the concentrations of upstream and downstream primers were 0.2, 0.4, 0.6, 0.8, and 1.0 μmol / L, respectively, and ddH2O was added to 10 μL. According to the instructions of 2× Rapid Taq Master Mix (Vazyme, catalog number: P222-02), the PCR reaction program was set as follows: 95°C for 3 min; 95°C for 15 s; 55°C for 15 s; 72°C for 45 s; 30 cycles; 72°C for 5 min. The results are shown in Figure 4 , Figure 4 Medium: DL 5000 DNAMarker; Lane 1: 0.2μmol / L; Lane 2: 0.4μmol / L; Lane 3: 0.6μmol / L; Lane 4: 0.8μmol / L; Lane 5: 1.0μmol / L. Figure 4 As shown, when the primer concentration was 0.4-0.8 μmol / L, the amplification effect was better.

[0065] 2. Using duck plague virulent DNA as template, optimize the number of cycles

[0066] According to the optimization results of primer concentration in step 1, the optimal primer amount was selected, and the number of cycles in the amplification program was set to 25, 30, and 35 cycles respectively, and the other conditions remained unchanged. Figure 5 , M in the figure: DL 5000 DNAMarker; Lane 1: 25 cycles; Lane 2: 30 cycles; Lane 3: 35 cycles. Figure 5 As shown, when the cycle number is 25-35, the amplification effect is better.

[0067] 3. Using duck plague virulent DNA as template, optimize the annealing temperature

[0068] According to the optimization results of primer concentration and cycle number in steps 1 and 2, the optimal primer concentration and optimal cycle number were selected, and the annealing temperature was set to 55-65℃ with a total of 8 temperature gradients, namely 55.0℃, 55.7℃, 56.9℃, 58.8℃, 61.1℃, 63.0℃, 64.3℃, and 65.0℃. Figure 6 In the figure, M: DL 5000 DNA Marker; Lane 1: 55.0℃; Lane 2: 55.7℃; Lane 3: 56.9℃; Lane 4: 58.8℃; Lane 5: 61.1℃; Lane 6: 63.0℃; Lane 7: 64.3℃; Lane 8: 65.0℃. Figure 6 As shown, when the annealing temperature is 55-63°C, the amplification effect is better.

[0069] 4. Optimization of duck plague attenuated virus PCR reaction conditions

[0070] Using duck plague attenuated DNA as template (concentration of 10 ng / μL), the primer concentration, cycle number and annealing temperature were optimized. The specific optimization steps refer to steps 1 to 3.

[0071] Figure 7 The results of primer concentration optimization show that the amplification effect is best when the primer concentration is 0.4 μmol / L. In the figure, M: DL2000 DNA Marker; Lane 1: 0.2 μmol / L; Lane 2: 0.4 μmol / L; Lane 3: 0.6 μmol / L; Lane 4: 0.8 μmol / L; Lane 5: 1.0 μmol / L.

[0072] Figure 8 The cycle number optimization result shows that the amplification effect is best when the cycle number is 35. In the figure, M: DL 2000 DNA Marker; Lane 1: 25 cycles; Lane 2: 30 cycles; Lane 3: 35 cycles.

[0073] Fig. 9 The results of annealing temperature optimization show that the amplification effect is best when the annealing temperature is 55.0℃. In the figure, M: DL 2000 DNA Marker; Lane 1: 55.0℃; Lane 2: 55.7℃; Lane 3: 56.9℃; Lane 4: 58.8℃; Lane 5: 61.1℃; Lane 6: 63.0℃; Lane 7: 64.3℃; Lane 8: 65.0℃

[0074] 5. Determination of PCR reaction conditions for identification of strong and weak duck plague viruses

[0075] (1) Determination of PCR reaction conditions for identification of strong and weak duck plague viruses

[0076] According to the results of the optimization of the PCR reaction conditions for strong and weak DPV, the best amplification effect can be obtained when the primer concentration is 0.4-0.8μmol / L, the annealing temperature is 55-63℃, and the number of cycles is 25-35 for strong DPV; the best amplification effect can be obtained when the primer concentration is 0.4μmol / L, the annealing temperature is 55℃, and the number of cycles is 35 for weak DPV. In order to ensure that both strong and weak DPV can achieve the best amplification effect when detecting unknown samples, the primer concentration (0.4μmol / L), annealing temperature (55℃) and number of cycles (35 cycles) with good amplification effects for both are selected as the optimal PCR reaction conditions for this method.

[0077] The final reaction system for the PCR method for identifying strong and weak duck plague viruses was determined to be: 2×Rapid Taq Master Mix 5μL, 0.4μmol / L of upstream and downstream primers each, 1μL of template, and ddH2O to make up to 10μL.

[0078] The reaction program was: 95°C for 3 min; 95°C for 15 s; 55°C for 15 s; 72°C for 45 s; 35 cycles; 72°C for 5 min.

[0079] (2) Amplification of mixed templates of strong and weak duck plague viruses

[0080] Under the determined optimal PCR reaction conditions, PCR amplification was performed on the mixed template of duck plague strong and weak toxins, and 5 μL of PCR product was taken for agarose gel electrophoresis. The results showed that the PCR method established by the present invention can well amplify the mixed samples of duck plague strong and weak toxins with different ratios. When the ratio of the template amount of duck plague strong to weak toxins is 1:3, the mixed template can obtain the best amplification effect (see Fig.10 , M in the figure: DL 5000 DNA Marker; Lane 1: 1 μL of strong DPV, 1 μL of weak DPV; Lane 2: 1 μL of strong DPV, 2 μL of weak DPV; Lane 3: 1 μL of strong DPV, 3 μL of weak DPV; Lane 4: 2 μL of strong DPV, 1 μL of weak DPV; Lane 5: 3 μL of strong DPV, 1 μL of weak DPV; Lane 6: 1 μL of strong DPV; Lane 7: 1 μL of weak DPV).

[0081] Example 4 Specificity Test

[0082] Using DPV CHv strain and DPV CVCC AV1222 strain as controls, the PCR method determined in Example 3 was used to perform PCR amplification on common pathogenic microorganisms that infect ducks, such as duck hepatitis B virus (DHBV), duck Tembusu virus (DTMUV), duck hepatitis A virus type 1 (DHAV-1), new duck reovirus (NDRV), Riemerella anatipestifer (RA), Pasteurella multocida (PM), and Salmonella (Sal.A). At the same time, a blank control was set, and 5 μL of PCR product was taken for agarose gel electrophoresis. The results showed that only the DPV CHv strong strain and the DPV CVCC AV1222 weak strain amplified bands of 2454 bp and 536 bp, respectively, and no specific amplification bands were found in other lanes, indicating that the PCR method established by the present invention has good specificity (see Fig.11In the figure, M: DL5000 DNA Marker; Lane 1: DPV CHv; Lane 2: DPV CVCC AV1222; Lane 3: DHBV; Lane 4: DTMUV; Lane 5: DHAV-1; Lane 6: NDRV; Lane 7: RA; Lane 8: PM; Lane 9: Sal.A; Lane 10: Mock).

[0083] Example 5 Sensitivity Test

[0084] 1. Sensitivity of PCR for identification of strong and weak duck plague viruses

[0085] The DNA of DPV CHv strain and DPV CVCC AV1222 strain were diluted to an initial concentration of 4.6 ng / μL, and then 10-fold gradient dilutions were performed for a total of 8 gradients (10 0 -10 -7 ). 1ul of gradient diluted DPV CHv strain and DPVCVCC AV1222 strain DNA were used as templates for PCR amplification to verify the sensitivity of strong and weak toxicity identification PCR. 5μL of PCR product was taken for agarose gel electrophoresis. The results showed that in the electrophoresis results of DPV CHv ( Fig.12 ), lanes 1-4 all amplified bands of 2454 bp in size, and lane 5 had a weaker band at the same position, indicating that the minimum sensitivity of this PCR method to DPV CHv strain DNA was 0.46 pg. The electrophoresis results of DPV CVCC AV1222 showed ( Fig.13 ), there were bands of 536 bp in lanes 1 and 2, and a weaker band appeared in the same position in lane 3, indicating that the minimum sensitivity of this PCR method to DPV CVCC AV1222 strain DNA was 46 pg.

[0086] Fig.12 Middle M: ​​DL 5000 DNA Marker; Lane 1: 4.6 ng; Lane 2: 460 pg; Lane 3: 46 pg; Lane 4: 4.6 pg; Lane 5: 0.46 pg; Lane 6: 46 fg; Lane 7: 4.6 fg; Lane 8: 0.46 fg.

[0087] Fig.13 Middle M: ​​DL 2000 DNA Marker; Lane 1: 4.6 ng; Lane 2: 460 pg; Lane 3: 46 pg; Lane 4: 4.6 pg; Lane 5: 0.46 pg; Lane 6: 46 fg; Lane 7: 4.6 fg; Lane 8: 0.46 fg.

[0088] 2. Sensitivity of national standard PCR to DPV CHv and AV1222 strains

[0089] The sensitivity verification method of the national standard PCR is the same as step 1. The test results show that the national standard PCR method can amplify at least 46fg of DPV CHv ( Fig.14 , Lane 6), the minimum amplified DNA content of DPV CVCC AV1222 strain was 0.46 pg ( Fig.15 , lane 5). This indicates that the sensitivity of the national standard PCR method to DPV CHv and DPV CVCC AV1222 strain DNA is 46fg and 0.46pg, respectively.

[0090] Fig.14 Middle M: ​​DL 2000 DNA Marker; Lane 1: 4.6 ng; Lane 2: 460 pg; Lane 3: 46 pg; Lane 4: 4.6 pg; Lane 5: 0.46 pg; Lane 6: 46 fg; Lane 7: 4.6 fg; Lane 8: 0.46 fg.

[0091] Fig.15 Middle M: ​​DL 2000 DNA Marker; Lane 1: 4.6 ng; Lane 2: 460 pg; Lane 3: 46 pg; Lane 4: 4.6 pg; Lane 5: 0.46 pg; Lane 6: 46 fg; Lane 7: 4.6 fg; Lane 8: 0.46 fg.

[0092] Example 6 Detection of cotton swab samples of artificially infected duck plague virulent and vaccine attenuated

[0093] 1. Artificial infection and oral and cloacal swab collection

[0094] (1) Artificial infection

[0095] Twenty one-day-old non-immunized ducklings were raised to 14 days of age and randomly divided into two groups, with 10 ducklings in each group. Each duckling in group 1 was intramuscularly injected with 1 mL of 1×10 3 TCID 50 / 100μL DPV CHv to establish a DPV virulent artificial infection model; each duckling in group 2 was intramuscularly injected with 1×10 4 ELD 50 The duck plague attenuated live vaccine (DPV CVCC AV1222) was used to establish an artificial infection model with the attenuated DPV vaccine.

[0096] Table 3 Establishment of artificial infection model

[0097]

[0098] (2) Oral and cloacal swab collection

[0099] The ducks in the above-mentioned groups 1 and 2 were randomly numbered from 1 to 10; at 6 and 12 hours (hpi) after strong virus infection and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 21, and 28 days (dpi) after infection, oral swabs and cloacal swabs of the ducks in group 1 were collected using sterile cotton swabs (a total of 300 samples); at 6 and 12 hours (hpi) after weak virus infection and 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days (dpi) after infection, oral swabs and cloacal swabs of the ducks in group 2 were collected using sterile cotton swabs (a total of 240 samples), and the heads of the cotton swabs were placed in 1.5 mL centrifuge tubes containing 0.5 mL PBS buffer and stored at -20°C.

[0100] 2. Extraction of viral DNA from cotton swabs

[0101] (1) Take out the 1.5 mL centrifuge tube containing the cotton swab from -20 °C and thaw it, centrifuge it at 8000 r / min for 5 min, transfer 250 μL of the supernatant to a new 1.5 mL centrifuge tube, add 25 μL of proteinase K, and shake to mix for 5 s;

[0102] (2) Add 250 μL of Buffer AL to the sample, invert 3-5 times, vortex mix for 15 seconds, and incubate in a 70°C water bath for 10 minutes;

[0103] (3) Add 250 μL of anhydrous ethanol to the sample, vortex mix for 15 seconds, and briefly centrifuge to collect droplets on the tube wall;

[0104] (4) Place the gDNA column into a collection tube, transfer the mixture from (3) into the column, and centrifuge at 10,000 rpm for 1 min;

[0105] (5) After repeating step (4) twice, discard the filtrate and transfer the gDNA column to a new collection tube;

[0106] (6) Add 500 μL of Buffer DW1 to the column, invert and mix twice, and centrifuge at 10,000 rpm for 1 min.

[0107] (7) Discard the filtrate, add 650 μL Buffer DW2 to the column, and centrifuge at 10,000 rpm for 1 min.

[0108] (8) After repeating step (7) once, discard the filtrate, centrifuge at 10000 r / min for 2 min, and dry at room temperature for 15 min;

[0109] (9) Transfer the column to a new 1.5 mL centrifuge tube, add 30 μL of Buffer AE preheated to 70°C to the center of the column membrane, let stand for 3 min, and centrifuge at 10,000 rpm for 1 min.

[0110] (10) The eluate is transferred back to the center of the column membrane for a second elution;

[0111] (11) Store the extracted DNA at -20°C.

[0112] 3. Oral swab test results of the artificially infected DPV CHv group

[0113] The strong and weak virus identification PCR method established by the present invention was used to detect 45 oral swabs of the artificially infected DPV CHv group, and a total of 14 positive samples were detected, with a detection rate of 31.1%; a total of 18 positive samples were detected by the national standard PCR method, with a detection rate of 40% ( Fig.16 , Table 4, Fig.16 Figure A in the middle shows the PCR test results for distinguishing strong and weak viruses; Figure B shows the national standard PCR test results).

[0114] Table 4 Detection rate of strong and weak virus identification PCR and national standard PCR for oral swabs of DPV CHv group

[0115]

[0116] 4. Cloacal swab test results of the artificially infected DPV CHv group

[0117] The strong and weak virus identification PCR method established by the present invention was used to detect 45 cloacal cavity swab DNAs of the artificially infected DPV CHv group, and a total of 20 duck plague strong virus samples were detected, with a detection rate of 44.4%. A total of 21 duck plague samples were detected by the national standard PCR method, with a detection rate of 46.7% ( Fig.17 , Table 5, Fig.17 Figure A in the middle shows the PCR test results for distinguishing strong and weak viruses; Figure B shows the national standard PCR test results).

[0118] Table 5 Detection rate of strong and weak virus identification PCR and national standard PCR for cloacal swabs of DPV CHv group

[0119]

[0120] 5. Detection of cotton swabs of duck plague vaccine immunization group

[0121] (1) Oral swab test results of duck plague vaccine immunization group

[0122] The strong and weak virus identification PCR method established by the present invention was used to detect 36 oral swab DNAs of the artificially infected DPV vaccine immunization group, and a total of 16 duck plague samples were detected, with a detection rate of 44.4%. A total of 19 duck plague samples were detected by the national standard PCR method, with a detection rate of 52.8%. ( Fig.18 , Table 6, Fig.18 Figure A in the middle shows the PCR test results for distinguishing strong and weak viruses; Figure B shows the national standard PCR test results).

[0123] Table 6 Detection rate of oral swabs of duck plague attenuated vaccine group by strong and weak virus identification PCR and national standard PCR

[0124]

[0125] (2) Cloaca swab test results of duck plague vaccine immunization group

[0126] The results of the test on the artificially infected DPV vaccine immunization group using the strong, weak and toxic PCR method established by the present invention showed that 28 duck plague weak toxins were detected in 36 cloacal swabs, with a detection rate of 77.8%. The national standard PCR method detected a total of 29 duck plague samples, with a detection rate of 80.6%. The positive detection rates of the two methods were comparable ( Fig.19 , Table 7, Fig.19 Figure A in the middle shows the PCR test results for distinguishing strong and weak viruses; Figure B shows the national standard PCR test results).

[0127] Table 7 Detection rate of strong and weak virus identification PCR and national standard PCR in cloacal swabs of duck plague attenuated vaccine group

[0128]

[0129] Example 7 Detection of Cloaca Swab Samples from Clinically Healthy Duck Farms

[0130] 1. Cloaca swab testing in Mianyang duck farm

[0131] According to the results of the artificial infection experiment, the cloaca sampling route with a higher detection rate was selected for the collection of clinical samples. The 8 cloaca swab samples from the Mianyang duck farm were tested using the strong and weak toxin identification PCR method established by the present invention. The test results showed that this method did not detect DPV DNA from the 8 samples. The test results of the national standard PCR were consistent with the strong and weak toxin identification PCR method established by the present invention, and no DPV DNA was detected. In the detection of cloaca swabs from the Mianyang duck farm, the detection rates of the strong and weak toxin identification PCR method and the national standard PCR method were both 0% ( Fig. 20 , 21 , Table 8, Fig. 20 Middle M: ​​DL 5000 DNAMarker; Lanes 1-8: Cloaca swab samples No. 1-8; Lane 9: DPV CHv; Lane 10: DPV CVCC AV1222; Lane 11: Blank control; Fig.21Middle: DL 2000 DNA Marker; Lanes 1-8: cloacal swab samples No. 1-8; Lane 9: DPVCHv; Lane 10: DPV CVCC AV1222; Lane 11: blank control).

[0132] Table 8 Detection rate of strong and weak virus identification PCR and national standard PCR in cloacal swabs of Mianyang duck farm

[0133]

[0134] 2. Testing of cotton swabs in Ya’an duck farm

[0135] The PCR method for distinguishing strong and weak toxins established by the present invention was used to test 40 cloacal swab samples from the Ya'an duck farm. The test results showed that DPV DNA was not detected in any of the 40 samples. The test results of the national standard PCR method were consistent with it, and no corresponding bands were amplified in all lanes. In the detection of cloacal swabs from the Ya'an duck farm, the detection rates of the PCR method for distinguishing strong and weak toxins and the national standard PCR method were both 0% ( Fig. 22 , 23 , Table 9, Fig. 22 Middle M: ​​DL 5000 DNA Marker; Lanes 1-40: Cloaca swab samples No. 1-40; Lane 41: DPV CHv; Lane 42: DPV CVCC AV1222; Lane 43: Blank control; Fig.23 Middle: DL 2000 DNA Marker; Lanes 1-40: cloacal swab samples No. 1-40; Lane 41: DPV CHv; Lane 42: DPV CVCC AV1222; Lane 43: blank control).

[0136] Table 9 Detection rate of strong and weak virus identification PCR and national standard PCR for cloacal swabs in Ya'an duck farm

[0137]

[0138] 3. Cloaca swab testing in Qionglai duck farm

[0139] (1) Swab sample testing at Qionglai No. 1 Duck Farm

[0140] The PCR method for distinguishing strong and weak toxins established by the present invention was used to test 24 cloacal swab samples from the first Qionglai duck farm. The test results showed that 10 DPV weak toxin samples were detected among the 24 samples. The national standard PCR method detected 18 samples containing DPV. In the test of cloacal swabs from Qionglai No. 1 duck farm, the detection rate of DPV weak toxin by the PCR method for distinguishing strong and weak toxins was 41.7%, and the detection rate of DPV by the national standard PCR method was 75% ( Fig.24 , 25, Table 10, Fig.24 Middle M: ​​DL 5000 DNAMarker; Lanes 1-24: Cloacal swab samples No. 1-24; Lane 25: DPV CHv; Lane 26: DPV CVCC AV1222; Lane 27: Blank control; Fig.25 Middle: DL 2000 DNA Marker; Lanes 1-24: cloacal swab samples No. 1-24; Lane 25: DPV CHv; Lane 26: DPV CVCC AV1222; Lane 27: blank control).

[0141] Table 10 Detection rate of strong and weak virus identification PCR and national standard PCR in cloacal swabs of No. 1 Qionglai duck farm

[0142]

[0143] (2) Swab sample testing at Qionglai No. 2 Duck Farm

[0144] The strong and weak toxin identification PCR method established by the present invention was used to test 44 cloacal swab samples from the second Qionglai duck farm, and a total of 5 swabs were found to contain DPV strong toxins and 18 swabs were found to contain DPV weak toxins. The test results of the national standard PCR method showed that 41 of the 44 swabs contained DPV. In the test of cloacal swabs from Qionglai No. 2 Duck Farm, the strong and weak toxin identification PCR method had a detection rate of 11.4% for strong DPV and 40.9% for weak DPV, while the national standard PCR method had a detection rate of 93.2% for DPV ( Fig.26 , 27 , Table 11, Fig.26 Middle M: ​​DL 5000 DNA Marker; Lanes 1-44: Cloaca swab samples 1-44; Lane 45: DPV CHv; Lane 46: DPV CVCC AV1222; Lane 47: Blank control, Fig. 27 Middle M: ​​DL2000 DNA Marker; Lanes 1-44: cloacal swab samples No. 1-44; Lane 45: DPV CHv; Lane 46: DPV CVCCAV1222; Lane 47: blank control).

[0145] Table 11 Detection rate of strong and weak virus identification PCR and national standard PCR in cloacal swabs of No. 2 Qionglai duck farm

[0146]

[0147] The PCR method for distinguishing strong and weak duck plague viruses based on the C-KCE LORF5 5'UTR region after duck plague splicing established in the present invention can effectively and accurately identify strong and weak duck plague strains, and can detect a minimum of 0.46 pg of strong DPV and 46 pg of weak DPV.

Claims

1. A primer for identifying strong and weak duck plague strains, characterized in that: The primer sequences are shown in SEQ ID NOs. 1 and 2.

2. A kit for identifying strong and weak duck plague strains, characterized in that: The kit comprises the primers according to claim 1.

3. A method for identifying strong and weak strains of duck plague for non-diagnostic or therapeutic purposes, characterized in that: The following steps are involved: (1) Extract DNA from the sample to be tested; (2) Use the primers described in claim 1 or the kit described in claim 2 to perform PCR detection, and judge whether it is a strong strain or a weak strain sample based on the fragment length; if the amplified fragment size is 2454bp, it is a strong strain sample; if the amplified fragment size is 525bp, it is a weak strain sample; among them, the strong strain is DPV CHv, and the weak strains are DPV CHa and DPV CVCCAV1222.

4. The method according to claim 3, characterized in that The PCR reaction system included 5 μL of 2×Rapid Taq MasterMix, 1 μL of 10 ng / μL template, and the concentrations of upstream and downstream primers were 0.4 μmol / L, respectively, and finally ddH2O was added to 10 μL.

5. The method according to claim 3, characterized in that: The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 45 s, for a total of 35 cycles; and finally extension at 72°C for 5 min.

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